Chemically Amplified Response Strategies for Medical Sciences

ABSTRACT

Compositions are provided in which dendrimers and/or nanoparticles are synthesized with multi-photon responsive elements and self-immolative oligomers. The compositions may be utilized to selectively deliver Payloads within tissue by irradiating the compositions. The compositions may also be used to amplify sensitivity to irradiation.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/244,886, filed on Sep. 23, 2009, which is hereby explicitlyincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a method for non-invasively inducingmulti-photon processes and releasing compounds of interest to localizedareas in living systems.

BACKGROUND OF THE INVENTION

The emerging technology of nanoparticle packaging offers a way topackage and deliver compounds of interest that offers significantadvantages, in some cases, to delivering certain types of payloads, suchas pharmaceuticals, antibodies, and labeling compounds. Manypharmaceutical agents are vulnerable to a reduction in efficacy due tosolubility and bioavailability problems. Nanoparticle packaging offers away to improve their effectiveness. By the appropriate design ofnanoparticles, the serum stability of pharmaceutical agents can beenhanced and solubility limitations bypassed.

Nanoparticles also offer the potential, at least, for targeted deliveryof their payloads to areas of specific interest. Frequently, an affinityreagent, such as an antibody attached externally to the nanoparticle, isused to direct the nanoparticle to its intended location.

A wide variety of nanoparticles are currently available and/or underdevelopment. One particular type of nanoparticle is the dendrimer (see,e.g., Cheng, Y., J. Wang, T. Rao, X. He, T. Xu, T.; “Pharmaceuticalapplications of dendrimers: promising nanocarriers for drug delivery”;Front. Biosci. 13 (2008) 1447-1471.)

Commercially available dendrimers include polyamidoamine (“PAMAM”)dendrimers and polypropylene imine (“PPI”) dendrimers. Somerepresentative examples of dendrimers and their uses are disclosed inU.S. Pat. Nos. 6,579,906, 6,570,031, 6,545,101, 6,506,218. 6,464,971,6,452.053, 6,410,680, 6,395,257, 6,365,562, 6,306,991, 6,288,253,6,228,978, 6,224,898, 6,187,897, 6,184,313, 6,113,946, 6,083,708,6,068,835, 5,990,089, 5,938,934, 5,902,863, 5,788,989, 5,736,346,5,714,166, 5,661,025, 5,648,186, 5,393.797, 5,393,795, 5,332,640,5,266,106, 5,256,516, 5,256,193, 5,098,475, 4,938,885 and 4,694,064.

Forming dendrimers into nanoparticles and/or microparticles, however,does not fully address the question of bioavailability of substancescarried by the dendrimers. With the rapid progress of nanotechnologyover the past decade, there is growing interest in polymericbiomaterials that can be remotely disassembled in a controlled fashionwith an external stimulus, but are otherwise stable under physiologicalconditions (Wang, W.; Alexander, C. Angew. Chem. Int. Ed., 2008, 47,7804-7806). Various internal and external stimuli, such as pH, specificenzymes, temperature, and ultrasound are being explored as releasemechanisms. (See, e.g., Murthy, N. X., M.; Schuck, S.; Kunisawa, J.;Shastri, N.; Frechet, J. M. J. Proc. Natl. Acad. Sci. U.S.A., 2003, 100,4995-5000; Veronese, F. M. S., O.; Pasut, G.; Mendichi, R.; Andersson,L.; Tsirk, A.; Ford, J.; Wu, G.; Kneller, S.; Davies, J.; Duncan, R.,Bioconjugate Chem., 2005, 16, 775-784; Chung, J. E. Y., M.; Yamato, M.;Aoyagi, T.; Sakurai, Y.; Okano, T. J. J., Controlled Release, 1999, 62,115-127; Liu, S. Q.; Tong, Y. W.; Yang, Y. Y., Biomaterials, 2005, 26,5064-5074; Na, K.; Lee, K. H.; Lee, D. H.; Bae, Y. H., Eur. J. Pharm.Sci., 2006, 27, 115-122; Gao, Z. G.; Fain, H. D.; Rapoport, N. J.,Controlled Release, 2005, 102, 203-222; Nelson, J. L.; Roeder, B. L.;Carmen, J. C.; Roloff, F.; and Pitt, W. G. Cancer Research 2002, 62,7280-7283).

One of these promising approaches is the use of light to trigger theremote disassembly of polymers (Goodwin, A. P.; Mynar, J. L.; Ma, Y. Z.;Fleming, G. R.; Frechet, J. M. J. J. Am. Chem. Soc. 2005, 127,9952-9953). Light stimulus is especially attractive as it can beremotely applied for a short period of time with high spatial andtemporal precision. Some forms of light, such as near-infrared (NIR)light, can penetrate deep into tissue and thus potentially have many invivo applications (see, e.g., Near-Infrared Applications inBiotechnology; Raghavachari, R., Ed.; Practical Spectroscopy Series 25;Marcel Dekker: New York, 2001).

Two-photon excitation microscopy, for example, has been used as analternative to confocal and deconvolution microscopy that providesdistinct advantages for three-dimensional imaging. In particular,two-photon excitation excels at imaging of living cells, especiallywithin intact tissues such as brain slices, embryos, intact organs, andeven entire animals. Two-photon excitation microscopy provides superioroptical sectioning at greater depths in thick specimens than is possibleby other methods. This ability to see within tissues demonstrates thepracticality of using two-photon technology for other purposes within atissue and/or organism.

Three-photon excitation is a related non-linear optical absorption eventthat can occur in a manner similar to two-photon excitation. Thedifference is that three photons must interact simultaneously with thefluorophore to illicit a transition to the excited singlet state. Abenefit of three-photon excitation is that successful absorptionrequires only a tenfold greater concentration of photons than two-photonabsorption, making this technique attractive for some experiments.

Multi-photon phenomena allow unparalleled spatio-temporal control, andwhere longer wavelengths are employed, also allow deeper penetrationinto turbid bulk media such as tissue. Despite the revolutionary impactthese phenomena have had on neuroscience, microscopy and lithography, ithas been generally very difficult to apply this technique in vivo tostimulate and/or deliver biomaterials, diagnostics, and/or drugs. Thetechnology for fully exploiting these advantages has lagged behind andthere is still an unmet need for biomaterials that can efficientlyrespond to light, especially NIR light. No robust systems currentlyexist for in vivo use of multi-photon-responsive materials to deliverPayloads of interest.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention features a composition thatcomprises a multi-photon responsive element covalently linked to aself-immolative backbone subunit. In various embodiments of this aspect,the multi-photon responsive element is a two-photon responsive element;non-limiting examples of which can be drawn from the bromo-coumaringroup, and other examples are described herein. In some embodiments ofthis aspect, the composition further comprises a molecular network, andmay further comprise a Payload. In various embodiments, the molecularnetwork may comprise acrylamide elements and/or PEG elements. In someembodiments the self-immolative backbone subunit is a self-immolativedendrimer oligomer, and/or may comprise an assembled dendriticstructure. Various possible self-immolative backbone subunits aredescribed herein.

In another aspect, the present invention features a kit containing acomposition of the invention.

In an additional aspect, the present invention features a method ofdelivering a Payload to a tissue, or a selected position, wherein acomposition of the invention as described herein, wherein thatcomposition comprises a Payload, is delivered to a tissue, or a selectedposition, and irradiated with an appropriate wavelength ofelectromagnetic radiation, e.g., light, so as to activate themulti-photon responsive element, which in turn disrupts the compositionof the invention in the selected tissue or in the selected location,thereby releasing the Payload. In some embodiments, the radiation usedis near infrared light, in other embodiments it may be UV light. Invarious aspects of the invention, the Payload may comprise apharmaceutical agent, stem cell differentiation factors, immunogens,and/or antibodies.

In a further aspect, the present invention features a method foramplifying sensitivity to electromagnetic radiation, or light, such asUV light or near infrared light. In some embodiments, this aspectfeatures a composition that comprises a multi-photon responsive elementand a self-immolative backbone. In some embodiments of this aspect, thiscomposition further features a molecular network, and may also feature apayload. In this aspect, the composition is then irradiated withelectromagnetic radiation, triggering the multi-photon responsiveelement together with the self-immolative backbone. In some embodimentsthat also have a molecular network and/or a Payload, this will disruptthe molecular network and/or release the Payload.

In still another aspect, the present invention features a method ofmanufacturing a composition of the invention as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an experimental set-up for demonstrating the highspatial and temporal resolution of multi-photon excitation (MPE). Thisphotograph shows fluorescence of a dye following one photon (above) andtwo-photon (below) excitation. Spatial and temporal stimuli control maybe realized by using two-photon radiation capable of penetrating bulkturbid media to a desired depth. Two-photon absorption (2PA) offersunparalleled 3D spatial and temporal control (Denk, W., Strickler, J. H.& Webb, W. W., 2-Photon Laser Scanning Fluorescence Microscopy, Science248, 73-76 (1990)). Using two intersecting beams of near infrared (NIR)laser light, pinpoint accuracy can be achieved. Activation will occuronly in regions of high NIR light intensity where the two beams cross,and does not happen outside the two beams to an appreciable degree.

FIG. 2 is a diagram illustrating self-immolative fragmentation ofdendrimers where the ovals represent the triggering group and thespheres represent possible Payloads.

FIG. 3 is a diagram illustrating self-immolative fragmentation of linearoligomers where the spheres represent the triggering group and thevertical ovals represent possible Payloads.

FIG. 4 is a diagram representing the structure of a second generationdendritic self-immolative crosslinker (in other words, a two-tiereddendritic self-immolative crosslinker).

FIG. 5 is a diagram representing inverse emulsion polymerization ofacrylamide assembled with self-immolative crosslinker 1 in the presenceof a Payload (e.g., enzymes, antibodies, toxins, and/or catalysts etc.)to form loaded nanoparticles.

FIG. 6 is a diagram representing the release of Payload triggered byIR-light.

FIG. 7 is a diagram representing synthesis of dendritic self-immolativecrosslinker 1, in which compound 8 is reacted with compound II to formthe first generation dendritic crosslinker Compound II is synthesized inone step from mono-protected PEG diamine with methyl acrloyl chloride.

FIG. 8 is a diagram representing the structure of a triggering group forself-immolative crosslinker 1.

FIG. 9 is a diagram representing the synthesis of a triggering group.

FIG. 10 is a diagram representing the structures of polyester compoundsthat unravel and fragment completely when one of the hydroxyl protectinggroups is cleaved by two-photon absorption.

FIG. 11 is a diagram representing a polymer strand (left) and itscomplete fragmentation (right).

FIG. 12A is a diagram representing emulsion condensation methods thatcan be used to form nanoparticle or microparticles encapsulating aPayload (hydrophilic or hydrophobic). FIG. 12B represents a multi-photonresponsive nanoparticle made from polymer strands that encapsulate aPayload and then unravel when directed by irradiation with two-photonlight. This concept may be thought of as an optical nanosyringe, capableof releasing a high concentration of Payload when and where directed bya practitioner.

FIG. 13 depicts (A) the degradation of polymer upon exposure to 350 nmlight for defined time periods in acetonitrile/H2O (9/1) and (B) GPCtraces of polymer 2 before irradiation (boxes), upon irradiation for 15min in acetronitrile/H20/Et₃N (9/0.5/0.5) (circles) and after 24 hrincubation at 37° C. after 15 min irradiation.

FIG. 14 depicts the degradation of polymer 2 in CH₃CN/H₂O (9/1)(circles) and in CH₃CN/H₂O/Et₃N (9/0.5/0.5) (boxes) after 15 min of UVirradiation followed by incubation at 37° C.

FIG. 15 depicts the degradation of polymer upon exposure to 350 nm lightfor defined time periods in acetonitrile/H₂O/Et₃N (9/0.5/0.5). The boxesrepresent 1 min of irradiation, circles 5 min, triangles 15 min.

FIG. 16 depicts the degradation of polymer upon exposure to 750 nm lightfor 5 hours in acetonitrile/H₂O (9/1) and incubation at 37° C.

FIG. 17 is an illustration of the formulation of nanoparticles, theirdegradation and light-triggered release of their encapsulated Nile Redpayload.

FIG. 18 depicts the fluorescence intensity of Nile Red encapsulatedwithin polymeric nanoparticles and upon irradiation with 350 nm light,monitored at 630 nm with excitation at 540 nm.

FIG. 19 depicts the fluorescence intensity of Nile Red encapsulatedwithin polymeric nanoparticles and after 20 min irradiation incrementswith 750 nm light, monitored at 630 nm with excitation at 540 nm.

FIG. 20 represents a polymer synthesized with a 4-bromo-coumarin (1 GM)multi-photon responsive element having increased efficiency.

FIG. 21 represents the structure of a self-immolative polymer.

FIG. 22 represents the steps involved in the synthesis ofself-immolative polymers bearing bromo-coumarin groups. This photoprotecting group is cleaved efficiently upon two photon irradiation with800 nm of light.

FIG. 23 represents the structure of a two-photon responsive element.

FIG. 24 represents the methods and steps used to synthesize acrosslinker comprising a multi-photon responsive element.

FIGS. 25 a-25 c represent data demonstrating the cleavage of atwo-photon responsive element in response to irradiation at 800 nm. InFIG. 25 a, the NMR profile of a multi-photon responsiveelement+crosslinker is depicted. FIGS. 25 b and 25 c show thecrosslinker after the multi-photon reaction followed by NMR and UV-Vis,respectively.

FIG. 26 reproduces photographs of hydrogels formulated by thecrosslinkers containing multi-photon responsive elements.

FIGS. 27 a and 27 b depict the results of assays showing the lowtoxicity of hydrogels formulated by the crosslinkers with multi-photonresponsive elements. The results depicted in FIG. 27 a indicate that ahydrogel synthesized with a multi-photon-responsive unit of the instantinvention is not toxic when the amount of cell growth in 3.5 days withno treatment is compared with the presence of the hydrogel with themulti-photon responsive element with and without irradiation, and aPEG-Acrylate hydrogel without a multi-photon responsive element (withand without radiation). All of these treatments showed growth whencompared with the negative control. The gel also showed little or notoxicity using the MTT-toxicity assay (comparing media alone, media plusirradiation, hydrogel, and hydrogel treated with irradiation. FIG. 27 bis a graph of cell viability using the MTT-toxicity assay.

FIG. 28 depicts a polyester backbone that will disassemble upondeprotection of its hydroxyl protecting group.

FIG. 29 depicts the method that is used to produce the self-immolativepolyester backbone.

FIG. 30 depicts several polyester and polycarbonate backbones that willdissemble upon removal of protecting groups that can be used with theinstant invention.

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

Various embodiments of the present invention provide methods, compoundsand formulations for packaging compounds and/or other substances (e.g.,Payloads) and for optical activation and/or release of these Payloads.In some embodiments of the present invention, methods, compounds, andformulations are used inside tissues and living organismsnon-invasively, with a previously unattainable control of depth andlocation. Some embodiments of the present invention also allow for theamplification of multi-photon driven processes so that a singlemulti-photon absorption event may produce multiple events in response,again with the ability to do so non-invasively and with a previouslyunattainable control of depth and location within turbid media such astissue. Such amplification may, for example, greatly increase anpractitioner's ability to conduct an assay within a tissue. Thepotential of the embodiments of the invention is large and broad,allowing previously invasive procedures to be performed non-invasively,and previously inaccessible target sites to be accessed specifically andprecisely for purposes such as treatment and/or diagnosis.

Some embodiments of the present invention provide polymers that arecapable of providing an amplified response to multi-photon irradiation.A multi-photon responsive moiety is repetitively embedded in a polymerduring or after the synthesis of that polymer. The polymer with themulti-photon responsive element may in turn be used in the formation ofmaterials, nanoparticles, and/or microparticles. When that multi-photonresponsive moiety simultaneously absorbs, for example, two photons,changes in that molecular moiety disintegrate the polymer and canconsequently cause a domino effect that unravels the entire material,nanoparticle, and/or microparticle. It is similar to a net where thecross-linking strands can be selectively removed from a distance,allowing what was trapped within the net to escape from the survivingstrands that can no longer, by themselves, enclose the former cargo.Thus, when multi-photon responsive moieties are incorporated intomaterials, nanoparticles and/or microparticles during their synthesis itestablishes sensitivity to multi-photon light stimulation, and this, inturn, allows the facile triggering of the fragmentation of thematerials, nanoparticles and/or microparticles at selected target sites.

Many embodiments of the present invention also incorporateself-immolative oligomers into the structure of the materials,nanoparticles and/or microparticles formed and used according to thepresent invention. These self-immolative oligomers are frequentlylinked, either directly or through other self-immolative oligomers, tothe multi-photon responsive elements. Thus, when the multi-photonresponsive element is triggered via the absorption of radiation, this inturn may activate changes to the self-immolative oligomers.

Some embodiments of the present invention provide the ability toactivate processes through turbid media, such as within an organism,non-invasively with a previously unattainable control of depth andlocation. This takes advantage of the ability of some forms of light,such as near infrared red light, to penetrate turbid media such astissue. In such embodiments of the present invention, when amulti-photon responsive element, repetitively built into the verystructure of a material, nanoparticle and/or microparticle,simultaneously absorbs multi-photon irradiation from an appropriatelight source, this activates changes in that multi-photon responsiveelement that disrupts the structure of that material, nanoparticleand/or microparticle causing it to virtually disintegrate at a precisetime and location chosen by the practitioner. This process may in turnactivate the Payload (e.g., changing the fluorescence profile of aPayload comprising a fluorescent molecule), or may allow the Payload toactivate a process taking place at the point of release (e.g., a Payloadcomprising an enzyme now capable of cleaving nearby substrates).

Some embodiments of the present invention provide an ability to producean amplified response to multi-photon-driven processes; such amplifiedresponses may be produced non-invasively, even beneath bulk turbidmedia, such as within the tissue of a living organism. Triggering themulti-photon responsive elements, with appropriate radiation, that areattached to self-immolative crosslinkers where both are incorporatedinto materials and/or polymers amplifies (or creates) their collectivesensitivity to light and allows for highly sensitive triggeredfragmentation at target sites. Materials and/or polymers that comprisesuch self-immolative crosslinkers together with multi-photon responsiveelements may in turn comprise part of nanoparticles and/ormicroparticles.

Some embodiments of the present invention provide self-immolativeoligomer and self-immolative oligomer strategies. Such oligomers and/orstrategies may be used in embodiments of the present invention to createhigher order molecular assemblies (bulk materials, nanoparticles and/ormicroparticles) with extreme sensitivity towards multi-photonirradiation. A single event—the cleavage of the triggering group—givesrise to multiple cleavages leading to significant disruption of thestructure, thus delivering the Payload at the time and location of thepractitioner's choosing. Thus, embodiments of the present inventionenable previously unattainable optical remote control and/or activationof materials, substances and processes.

FIG. 10 illustrates examples of some polyesters that will unravel andfragment completely when one of the hydroxyl protecting groups iscleaved by two-photon absorption.

Novel self-immolative oligomers, by themselves, may also be featured asembodiments of the present invention.

Materials, nanoparticles, and/or microparticles synthesized according tothe instant invention may incorporate only self-immolative oligomersdescribed in the present invention, or they may incorporate any suitableself-immolative oligomers.

Earlier work using light-activated self-immolative oligomers has beenreported. Shabat described use of an ortho-nitrobenzyl group as alight-sensitive trigger in “Self-Immolative Dendrimers” (2003)Angewandte Chemie-International Edition, 42, 4494-99.

Previous work developing self-immolative oligomers, albeit not ones thatare light-sensitive, has produced dendritic structures which can bebranched (FIG. 2) or linear (FIG. 3) (see, generally, Amir, R. J.,Danieli, E. & Shabat, D.; “Receiver-amplifier, self-immolative dendriticdevice”; (2007) Chemistry-a European Journal 13, 812-21; Amir, R. J.,Popkov, M., Lerner, R. A., Barbas, C. E. & Shabat, D.; “Prodrugactivation gated by a molecular “OR” logic trigger;” (2005) AngewandteChemie-International Edition 44, 4378-81; Amir, R. J. & Shabat, D.;“Self-immolative dendrimer biodegradability by multi-enzymatictriggering”; (2004) Chemical Communications, 1614-15; Amir, R. J. &Shabat, D.; “Domino dendrimers” (2006) Polymer Therapeutics I: Polymersas Drugs, Conjugates and Gene Delivery Systems 192, 59-94; Weinstain,R., Sagi, A., Karton, N. & Shabat, D.; “Self-immolative comb-polymers:Multiple-release of side-reporters by a single stimulus event”; (2008)Chemistry-a European Journal 14, 6857-6861; Sagi, A., Weinstain, R.,Karton, N. & Shabat, D.; “Self-immolative polymers”; J Am Chem. Soc(2008) 130, 5434; Amir, R. J., Shabat, D; U.S. Patent Publication No. US2006/0269480). These dendrimers are well-defined syntheticmacromolecules characterized by a large number of functional groups.Their well-defined structure distinguishes them from many other classesof nanoparticles and/or microparticles.

Generally speaking, dendrimers are molecules synthesized with repeatingunits radiating out from a core. Each tier of repeating units isreferred as a “generation”. This process, which is classified as“convergent” or “divergent”, creates highly branched macromoleculesemanating from the central core. Divergent dendrimers are synthesizedoutward from the central core. Dendrimers can be made in a wide varietyof sizes and molecular weights, and can include inner cavities that maybe exploited to carry molecules of interest. Dendrimers can also beadapted so as to have uniform or discrete functionalities, and can befurther adapted with regard to surface moieties, interior moieties, andsolvent interactions.

Dendritic macromolecules may also be used for the amplification of themolecular effects following chemical or enzymatic triggering. Asecond-generation dendrimer (in other words, a dendrimer with two tiersof oligomers) incorporating 2,6-bis(hydroxymethyl)-p-cresol units andcarbamate linkages has been synthesized and demonstrated to undergotriggered disassembly (see, e.g., FIG. 2). Cleavage of the triggeringgroup, frequently by means of an enzyme, initiates a spontaneouscyclization to form an N,N′-dimethylurea derivative. The resultantphenol then undergoes a double 1,4-quinone methide rearrangement,followed by spontaneous decarboxylation. The cyclization/rearrangementsteps are then repeated to liberate four pyrene reporter molecules. Athird-generation self-immolative dendrimer of the same general structurebearing eight 4-nitroaniline reporter molecules was also successfullysynthesized. Thus, higher generation self-immolative dendrimers providehigher signal-to output ratio. Consequently, a single event—cleavage ofthe triggering group—gives rise to multiple outputs, and thesignal-to-output ratio may be tuned by controlling the degree ofbranching.

Thus, in certain embodiments of the present invention, suchself-immolative strategies may be used to create higher order molecularassemblies (materials, nanoparticles and microparticles) with enhancedsensitivity towards multi-photon irradiation. A single event—thecleavage of the triggering group—gives rise to multiple cleavagesleading to the disintegration of the overall structure with itsassociated network. Self-immolative crosslinkers used to crosslink thestrands of a molecular network facilitate the rapid and controlleddisintegration of the a molecular network.

In some aspects of the embodiments of the present invention, thestructure that incorporates the self-immolative oligomers comprises adendritic structure.

Some embodiments of the present invention provide methods forsynthesizing and utilizing nanoparticles and/or microparticles thatinclude Payload(s) whereby the packaged Payload(s) can be released fromthe polymeric structure of the nanoparticles and/or microparticles uponreceipt of an optical signal, usually a multi-photon stimulation, whichcauses the self-immolative disruption of the particle's molecularnetwork, thereby releasing the Payload(s). By thus creating a molecularnetwork that can be released remotely using wavelengths of light capableof penetrating tissue, compounds included in the Payload may beactivated and/or released non-invasively inside living systems at thepractitioner's discretion, with a previously unattainable control ofdepth and location. The potential of such remote control is large andbroad, allowing previously invasive procedures to be performednon-invasively, and previously inaccessible target sites to be reachedspecifically and precisely for treatment and/or diagnosis.

Embodiments of the present invention provide a broadly-faceted strategyfor the delivery of biological agents, i.e., Payload(s), viananoparticles and/or microparticles involve sequestration orencapsulation the Payload(s) within the nanoparticles and/ormicroparticles. Before the teachings of the present invention, the typesof small particles frequently involved in this approach were nanogels,microgels, nanospheres, microspheres, polymer micelles, and/orpolymerized liposomes. In general, retention of the active compound inthe nanoparticles was achieved by physical entrapment or bythermodynamic forces such as hydrophobic interactions. The use ofmicrobeads with a slowly degradable shell, consisting of, for example,poly(beta-amino ester)s or poly(ortho ester) is described in theliterature (Wang, C., Ge, Q., Ting, D., Nguyen, D., Shen, H.-R., Chen,J., Eisen, H. N., Heller, J., Langer, R. & Putnam, D.; “Molecularlyengineered poly(ortho ester) microspheres for enhanced delivery of DNAvaccines”; Nature Materials (2004) 3, 190-196). Such microspheres wereconveniently prepared from a pre-formed polymer using a water-oil-waterdouble emulsion method. Similarly, a number of pre-formed hydrophobicpolymers are used to encapsulate hydrophobic drugs or hydrophilicmacromolecules in processes known as nanoprecipitation and doubleemulsion condensation (Bilati, U., Allemann, E. & Doelker, E.;“Nanoprecipitation versus emulsion-based techniques for theencapsulation of proteins into biodegradable nanoparticles andprocess-related stability issues;” Aaps Pharmscitech (2005) 6,E594-604). The teachings of the present invention are in contrast to thesmall particles thus described and improve upon such particles bycombining multi-photon responsive units and self-immolative oligomers indefined structures that provide multi-photon control to molecularnetworks that encapsulate Payload(s).

Some embodiments of the present invention therefore feature selectedconcepts drawn from self-immolative dendritic structures are combinedwith unstructured polymers to provide nanoparticles and/ormicroparticles that that can be dissembled rapidly, but only whentriggered by multi-photon irradiation. Some embodiments of the presentinvention improves upon the construction of self-immolative dendrimerssuch as those described by Shabat et al. (see supra) by further addingpolymers to mechanically entrap multiple copies of molecules within thenanoparticle and/or microparticle, as opposed to each Payload moleculebeing covalently coupled to a multi-photon responsive moiety.

In some embodiments of the present invention, self-immolative dendriticcrosslinkers comprising multi-photon responsive elements are used in theinverse emulsion polymerization of acrylamide to form a molecularnetwork forming nanoparticles and/or microparticles. Such nanoparticlesand/or microparticles may also be designed to comprise Payload(s), andmay also contain a Payload(s). The final steps of assembly of thenanoparticle in many embodiments may take place in the simultaneouspresence of coupled multi-photon responsive elements and self-immolativeoligomers, the Payload(s), and the constituents of the molecularnetwork, e.g., acrylamide. It is one advantage of some embodiments ofthe present invention that the final assembly of the nanoparticle and/ormicroparticles may take place in conditions that should not denature orotherwise disrupt the Payload(s)'s constituents. It is also an advantageof some embodiments of the present invention that the Payload(s) is notcovalently linked to any part of the molecular network in which it iscontained.

A significant advantage of the present invention is that reliance on amechanical entrapment mechanism relieves the practitioner of the need totailor the chemistry of a selected multi-photon compound to each andevery Payload desired. This enables a tremendously versatile strategyfor Payload delivery.

In other embodiments of the invention, polymers other than acrylamidecan be attached to the self-immolative dendritic crosslinkers andmulti-photon responsive elements of the present invention. One suchpolymer is Acetalated-dextran. Another example of a non-acrylamidenetwork that may be used is polyethylene glycol (PEG).

For some embodiments of the present invention, multi-photon responsiveelements with high two-photon action cross sections are preferred,generally about 1 GM (Goeppert-Mayer, GM; 1 GM=10-50 cm⁴·s/photon).

Photo protecting groups (PPGs) were originally developed as protectinggroups for organic synthesis. Today, however, PPGs span an entire gamutof applications from release of neurotransmitters, ions, drugs,biomolecules like proteins and enzymes¹⁷ to commercial chemicals such asfragrances (Miranda, K. M., Nagasawa, H. T. & Toscano, J. P.; “Donors ofHNO”; Current Topics in Medicinal Chemistry (2005) 5, 647-664; Pavlos,C. M., Xu, H. & Toscano, J. P. “Photosensitive precursors to nitricoxide”; Current Topics in Medicinal Chemistry (2005) 5, 635-645; Specht,A., Ziarelli, F., Bernard, P., Goeldner, M. & Peng, L.; “para-Sulfonatedcalixarenes used as synthetic receptors for complexing photolabilecholinergic ligand;” Helvetica Chimica Acta (2005) 88, 2641-2653;Herrmann, A.; “Controlled release of volatiles under mild reactionconditions: From nature to everyday products”; AngewandteChemie-International Edition (2007) 46, 5836-5863). The choice of whichPPG to use depends on the system under consideration, and must becustomized to each specific application. There has been extensiveresearch into development of one photon PPGs (OP-PPGs) and inapplications of these OP-PPGs to release molecules of interest (Millar,A. G., Zucker, R. S., Ellis-Davies, G. C. R., Charlton, M. P. & Atwood,H. L.; “Calcium sensitivity of neurotransmitter release differs atphasic and tonic synapses;” Journal of Neuroscience (2005) 25,3113-3125; Falvey, D. E. & Sundararajan, C.; “Photoremovable protectinggroups based on electron transfer chemistry”; Photochemical &Photobiological Sciences (2004) 3, 831-838; Corrie, J. E. T. & Barth,A.; “Decarboxylation is a major pathway in photolysis of caged calciumreagents”; Biophysical Journal (2005) 88, 340a-340a). However,developments in two-photon PPGs (TP-PPGs) are still in their infancy.Despite the scarcity of TP-PPGs, their applications are manifold inbiological sciences (Furuta, T., Wang, S. S. H., Dantzker, J. L., Dore,T. M., Bybee, W. J., Callaway, E. M., Denk, W. & Tsien, R. Y.;“Brominated 7-hydroxycoumarin-4-ylmethyls: Photolabile protecting groupswith biologically useful cross-sections for two photon photolysis”;Proc.s Natl. Acad. Sci. USA (1999) 96, 1193-200; Tsien, R. Y., Miyawaki,A., Zacharias, D., Lev-Ram, V., Llopis, J., Baird, G., Zlokarnik, G.,Li, W. H., Furuta, T. & Dore, T.; “New molecules to peek and poke atsignal transduction”; Jou'rnal of General Physiology (1999) 114, 3A-3A;Aujard, I., Benbrahim, C., Gouget, M., Ruel, O., Baudin, J. B., Neveu,P. & Jullien, L.; “o-Nitrobenzyl photolabile protecting groups withred-shifted absorption: Syntheses and uncaging cross-sections for one-and two-photon excitation”; Chemistry-a European Journal (2006) 12,6865-6879). One attractive aspect of using Near Infrared radiation isits ability to penetrate and propagate deep into turbid media; however,in spite of that prior applications of TP-PPGs have focused on the usein thin layer settings.

One non-limiting example of a near infrared-labile protecting group thatcan be used in embodiments of the present invention is thebromo-coumarin class; a well-established class of two-photon protectinggroups (see, e.g., Furuta, T., Watanabe, T., Tanabe, S., Sakyo, J. &Matsuba, C. Phototriggers for nucleobases with improved photochemicalproperties. Organic Letters (2007) 9, 4717-4720). Such compounds have aproven track record of success when used as a two-photon photoremovableprotective group in the case of nucleobases (Almutairi, A., Rossin, R.,Shokeen, M., Hagooly, A., Ananth, A., Capoccia, B., Guillaudeu, S.,Abendschein, D., Anderson, C. J., Welch, M. J. & Frechet, J. M. J.;“Biodegradable dendritic positron-emitting nanoprobes for thenoninvasive imaging of angiogenesis”; Proc. Natl. Acad. Sci. USA (2009)106, 685-690) and in the case of uncaging glutamate in brain cells(Almutairi, A., Guillaudeu, S. J., Berezin, M. Y., Achilefu, S. &Frechet, J. M. J.; “Biodegradable pH-sensing dendritic nanoprobes fornear-infrared fluorescence lifetime and intensity imaging;” J Am ChemSoc; (2008) 130, 444).

Bromo-coumarins have been used synthesize dendritic structures for usewith some embodiments of the present invention. When in its carbamateform (FIG. 8), bromo-coumarin has been shown to release amines upontwo-photon IR photolysis with a high action cross-section (0.95 GM at740 nm and 0.37 GM at 800 nm) (Almutairi et al., J Am Chem. Soc (2008)).

The synthesis of a compound useful as a multi-photon responsive elementis depicted in FIG. 9, compound 13. It can be accessed from commerciallyavailable 4-bromoresorcinol 14 by reacting it with ethyl4-chloroacetoacetate to give compound 15. Hydrolysis of 15 will affordthe desired compound 13. In order to install the coumarin triggeringgroup onto dendritic crosslinker 1, compound 13 was converted intocompound 16 by reacting it with p-nitrophenyl-chloroformate.

A number of other promising two-photon-protecting groups with highaction cross-sections (1-6 GM) are currently available that can be usedwith the present invention (see, e.g., Gug, S., Bolze, F., Specht, A.,Bourgogne, C., Goeldner, M. & Nicoud, J. F.; Molecular Engineering ofPhotoremovable Protecting Groups for Two-Photon Uncaging; AngewandteChemie-International Edition (2008) 47, 9525-9529).

Other two-photon systems with large two-photon-action cross-sectionsused in neuroscience, fluorescence microscopy, cell biology, andphotodynamic therapy have been investigated for the covalent release ofbiomolecules. In previous studies, however, the molecule of interest(i.e., the Payload(s)) was directly and covalently linked to thestructure containing the two-photon moiety.

Embodiments of the present invention improve upon that art by entrappingmultiple copies of the Payload(s) within a nanoparticle and/ormicroparticle by effectively surrounding them with a net built frompolymers, but allowing the covalently photo-unraveling of this net at asite and a time of the practitioner's choosing. Because the molecularstructure of the molecular network is built upon self-immolativeprinciples, the effect of a single multi-photon absorption event causesthe fragmentation of more than one bond, and thus the self-immolativestructure amplifies the overall effect of the absorption event.

In some embodiments that use multi-photon responsive moieties,two-photon responsive moieties are used. By requiring multi-photonstimulation, or two-photon stimulation, an added element of control hasbeen added to the system that may require high local intensities at aspecific location with negligible release elsewhere.

In some embodiments of the present invention, multi-photon responsiveelements coupled to self-immolative dendritic crosslinkers, such as theexemplary structure shown in FIG. 4, improve over what existed prior tothe present invention. Self-immolative linkers such as shown in FIG. 4may further be used in the inverse emulsion polymerization of acrylamide(FIG. 5) to form novel nanoparticles and/or microparticles. Thus, thepresent invention improves upon prior forms of hydrogel nanoparticles(e.g., Cohen, J. A., Beaudette, T. T., Tseng, W. W., Bachelder, E. M.,Mende, I., Engleman, E. G. & Frechet, J. M. J. T-Cell Activation byAntigen-Loaded pH-Sensitive Hydrogel Particles in Vivo: The Effect ofParticle Size. Bioconjugate Chemistry 20, 111-119 (2009); Cohen, J. L.,Almutairi, A., Cohen, J. A., Bernstein, M., Brody, S. L., Schuster, D.P. & Frechet, J. M. J. Enhanced cell penetration of acid-degradableparticles functionalized with cell-penetrating peptides. BioconjugateChemistry 19, 876-881 (2008)). In some embodiments of the presentinvention, acrylamide groups capable of being polymerized are introducedand connected to the core of the crosslinker comprising multi-photonresponsive elements and self-immolative crosslinkers throughpolyethylene glycol (“PEG”) chains. The PEG chains may also be modifiedfor improved water solubility and compatibility with inverse emulsionpolymerization methods.

Referring to FIGS. 4 and 5, inverse emulsion copolymerization ofacrylamide and crosslinker 1 functionalized with an acrylamido group inthe presence of a water soluble Payload (e.g. an enzyme or an APA)resulted in the formation of self-immolative nanoparticles with anentrapped Payload (FIG. 5).

In conventional dendrimers, one triggering event may lead to thecleavage of one bond (signal-to-output ratio equals 1). In someembodiments of the present invention, a single triggering event willlead to the number of cleaved bonds equal to the number of branches inthe case of dendritic crosslinker, or to the number of monomer units inthe case of linear polymeric crosslinker. Irradiation with IR light maybe thus used to trigger widespread fragmentation of the crosslinkerswithin the microparticle, resulting in “swelling” of the microparticleand subsequent release of its payload (FIG. 6).

Systems, such as those of the present invention, capable of a collectiveand amplified response have not been heretofore developed utilizing thefeatures of multi-photon response coupled to self-immolative oligomers,together with an ability to facilely package Payloads of interest to apractitioner.

Thus, according to the present invention, procedures are described toprovide materials that have an amplified, highly sensitive, response tonon-invasive multi-photon irradiation. One approach of the instantinvention is to incorporate self-immolative oligomers as crosslinkersinto materials, nanoparticles and/or microparticles so as to amplifytheir sensitivity to light and allow for highly sensitive and triggeredfragmentation at targeted sites. When multi-photon responsive elementsare incorporated, together with self-immolative oligomers, within amaterial, nanoparticle and/or microparticle in a specific manner, theycan initiate a cascade of changes within the material, nanoparticleand/or microparticle when triggered by appropriate irradiation. Thisstrategy enables optical and remote control and/or activation ofmaterials, substances and processes (depending on the Payload, whichmight comprise an enzyme, catalyst, luminescent molecule, etc.) withpreviously unattainable control, especially in regard to penetrationdepth. The multi-photon responsive elements and self-immolativeoligomers may form a polymeric structure similar to a backbone, asdepicted in FIG. 3, or they may be used in a dendritic structure, suchas in FIG. 2. In another approach multi-photon responsive elements aredirectly incorporated within the polymeric backbone of materials whereinthey are able to absorb two photons simultaneously to initiate a cascadeof changes within a higher order assembly.

Both the backbone form and the dendritic form, or combinations thereof,may be, in turn, used as crosslinkers in a molecular network in thepresent invention.

Embodiments of the present invention further provide for the activationof processes within an organism brought about by the release ofPayload(s) non-invasively, with a previously unattainable control ofdepth and location. In some embodiments of the present invention, when asingle responsive molecular unit, repetitively built into the molecularnetwork of a material, nanoparticle and/or microparticle simultaneouslyabsorbs two photons, the changes in that molecular unit cause themolecular network of the material, nanoparticle and/or microparticle tovirtually disintegrate at a precise time and location chosen by thepractitioner of the invention, brought about by the destruction of thecrosslinkers.

Additionally, embodiments of the present invention also provide polymersas well as materials, nanoparticles and/or microparticles that have anamplified response to multi-photon irradiation. In a non-limitingexample, when a single responsive molecular unit that is repetitivelyembedded in a polymer, such as one used to construct a material,nanoparticle, and/or microparticle, simultaneously absorbs two photons,changes in that molecular unit cause a domino effect that unravels theentire material as a whole. The effect of a single two-photon absorptionis amplified throughout the molecular network because it triggers theself-immolative property of the molecular network leading to thenetworks disintegration. Thus, a single event, the absorption oftwo-photon irradiation can produce multiple events resulting in therelease of a Payload(s), and the release of the Payload(s), in turn, mayalso trigger multiple events.

Embodiments of the present invention may produce an amplified responseto two-photon-driven processes non-invasively, even beneath bulk turbidmedia, such as within the tissue of a living organism. This inventionenables previously unattainable optical remote control and/or activationof materials, substances and processes. Even if the two-photonirradiation is attenuated, the amplification inherent in the presentinvention allows functioning of present invention in spite of thisattenuation.

In some embodiments of the present invention, the collective responsefrom the Payload(s) also amplifies the desired effect, making the entiresystem many times more sensitive. Thus, in the art, where a practitionermay have been forced to search for a small number of single two-photonabsorption events, in embodiments of the present invention, by carefulselection of appropriate Payload(s), may amplify the result. Thus, whenthe absorption of multi-photon irradiation causing the disruption of amolecular network fabricated according to the teachings of the presentinvention releases a Payload(s), the Payload(s) may in turn triggerprocesses or events.

In a non-limiting example, a combination of nanoparticles may be usedwhere the Payload(s) in one set may comprise a luminescent enzyme andthe Payload(s) in the other set may comprise the enzyme's substrate. Asmall number of multi-photon absorption events may then result in asignificant burst of luminescence as the enzyme and substrate areunited.

In another non-limiting example, the Payload(s) can consist ofantibodies. In this case, a single multi-photon absorption event canrelease multiple antibodies capable of binding multiple antigens at thepoint of release. This is in sharp contrast to the case where a singletwo-photon label attached to an antibody releases only a singleantibody.

Yet another example is Payload(s) consisting of small moleculeanticancer drugs. See, e.g., Schoell, I., G. Boltz-Nitulescu, and E.Jensen-Jarolim. 2005, “Review of novel particulate antigen deliverysystems with special focus on treatment of type I allergy”, Journal ofControlled Release 104:1-27.

According to embodiments of the present invention, materials and/ormolecular networks are provided that have an amplified, and thus highlysensitive response, to non-invasive multi-photon irradiation. Oneapproach of the instant invention is to incorporate self-immolativecrosslinkers into materials that comprise the network formingnanoparticles and/or microparticles so as to amplify their sensitivityto light and allow for highly sensitive and triggered fragmentation attargeted sites. When multi-photon responsive molecular units areincorporated within a material, nanoparticle and/or microparticle insuch a manner, they can initiate a cascade of changes within thematerial, nanoparticle and/or microparticle when triggered, disruptingthe structure of the material, nanoparticle and/or microparticle. Thisstrategy enables optical and remote control and/or activation ofmaterials, substances and processes (depending on the Payload, e.g., anenzyme, a catalyst, an APA, etc.) with previously unattainable control,especially in regard to penetration depth, as well as a response that isamplified with respect to the multi-photon absorption event.

The instant inventive methods and compounds may be applied towardsnon-invasive surgery, drug delivery, diagnostic techniques, andnon-invasive suture removal as well as other techniques such asinjectable implants that harden upon irradiation with two photons. Whenself-immolative systems coupled to multi-photon responsive elements areused as crosslinkers in a nanoscale or microscale hydrogel loaded withbioactive agents, the release and activity Payload (e.g. enzymes) willbe triggered by absorption of IR-light. By forming a cage around thePayload(s) that can be uncoupled remotely, the present inventionprovides a ‘locate and release’ technology enabling the effectivedelivery of a cargo encapsulated in nanoparticles or microparticles tospecific areas.

The instant invention overcomes logistical issues that have heretoforeincreased the difficulties inherent with commercialization of many drugdelivery systems, such as serum instability and short storage life. Theinstant invention provides the pharmaceutical worker in the art withmethods to deliver drugs in a more accurate and more cost-effectivemanner as well as providing means for evaluating their performance.

The instant invention provides advantages in the diagnostics arena whereit can be used to enhance the early diagnosis of disease, which, inturn, improves the possibility of successful treatment. While two-photonimaging, because of its inherent penetrating power, could be useddirectly to image events and conditions within tissues; the presentinvention enhances to capabilities of the technique. By providing annon-linear and amplified response, the present invention enhances thepower of imaging using two-photon imaging. If the nanoparticle and/ormicroparticle of the invention is localized by affinity to an area ofinterest, e.g., a metastatic tumor's location, the amplified response ofthe present invention increases the ease of detection.

These applications open to the present invention are facilitated by theability of the present invention to easily tune the size, chemistry,topology, and the biological response of these materials throughchemical design, synthesis and engineering. Encapsulation of viralparticles, DNA, proteins, and adjuvants, in the same nanoparticle mayhelp to realize optimal cytotoxic T lymphocytes (CTLs) responses andantibody responses by sequestering the vaccine components until theyreach the target cell, delivering them to a particular class of cells,and/or aiding in their presentation via the desired pathway. Moreover,with the development of new adjuvants, gene based vaccines and othermulti-modal approaches to vaccination, it becomes increasingly importantto use carriers tailored to these novel immunization approaches.

It should be appreciated that certain features of the present inventionthat are, for clarity reasons only, described in the context of separateembodiments, may also be provided as part of the present invention incombination in a single embodiment. On the other hand, various featuresof the invention, which are, for clarity purposes only, described in thecontext of a single embodiment. may also be provided separately or inany suitable subcombination.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

The invention illustratively described herein can suitably be practicedin the absence of any element or elements, limitation or limitationsthat is not specifically disclosed herein. Thus, for example, the terms“comprising,” “including,” “containing,” etc. shall be read expansivelyand without limitation. Furthermore, although elements of the presentinvention has been defined broadly with the compositions and/or methodsof the invention comprising a variety of elements, in particularembodiments, individual elements may be claimed as “consisting of” or“consisting essentially of” that individual element.

Additionally, the terms and expressions employed herein have been usedas terms of description and not of limitation, and there is no intentionin the use of such terms and expressions of excluding any equivalent ofthe invention shown or portion thereof, but it is recognized thatvarious modifications are possible within the scope of the inventionclaimed. Thus, it should be understood that although the presentinvention has been specifically disclosed by preferred embodiments andoptional features, modifications and variations of the inventionsembodied herein disclosed can be readily made by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of the inventions disclosed herein. The inventions havebeen described broadly and generically herein. Each of the narrowerspecies and subgeneric groupings falling within the generic disclosurealso form the part of these inventions. This includes within the genericdescription of each of the inventions a proviso or negative limitationthat will allow removing any subject matter from the genus, regardlessor whether or not the material to be removed was specifically recited.In addition, where features or aspects of an invention are described interms of the Markush group, those schooled in the art will recognizethat the invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group. Further, when areference to an aspect of the invention lists a range of individualmembers, as for a non-limiting example, ‘the letters A through F,inclusive,’ it is intended to be equivalent to listing every member ofthe list individually, that is ‘A, B, C, D, E and/or F,’ andadditionally it should be understood that every individual member may beexcluded or included in the claim individually. Additionally, when areference to an aspect of the invention lists a range of individualnumbers, as for a non-limiting example, ‘0.25% to 0.35%, inclusive,’ itis intended to be equivalent to listing every number in the rangeindividually, and additionally it should be understood that any givennumber within the range may be included in the claim individually.

The steps depicted and/or used in methods herein may be performed in adifferent order than as depicted and/or stated. The steps are merelyexemplary of the order these steps may occur. The steps may occur in anyorder that is desired such that it still performs the goals of theclaimed invention.

Furthermore, although the present invention has been described inconjunction with specific embodiments thereof, it is evident that manymodifications, alternatives, and variations will be apparent to thoseskilled in the art. Accordingly, the present invention should beconstrued to embrace all such modifications, alternatives, andvariations that fall within the spirit and broad scope of the claims.

All publications, patents, and patent applications mentioned in thisspecification are hereby incorporated in their entirety by referenceinto the specification to the same extent as if each individualpublication, patent, or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention.

DEFINITIONS

As used herein, the term “Payload”, or “Payload(s)”, refers to anycompound of interest that can be incorporated into the molecular networkof the present invention. Non-limiting examples of Payloads comprisechemicals such as drugs, APAs, pharmaceutical agents, and/or radioactiveelements; a Payload can also comprise proteins such as antibodies,antibody fragments, antigens, cytokines; a Payload can also comprisenucleic acids, including DNAs, RNAs, siRNAs, antisense oligonucleotides;a Payload can also comprise detectable labels, such as fluorescentcompounds (e.g., rhodamine dyes, near infrared fluorescent agents, orfluorescent proteins) and MRI contrast agents; and/or a Payload can alsocomprise a cocktail that comprises more than one compound (e.g., apharmaceutical agent and an antibody). In the embodiment where a Payloadconsists of multiple entities, each may be present separately within thenanoparticle and/or microparticle or some of the entities may beconjugated together in various combinations. Furthermore the multipleentities may be present in separate particles and the particle may becombined for use. The Payload(s) delivered by the embodiments of thepresent invention may routinely contain pharmaceutically acceptableconcentrations of salts, buffering agents, preservatives, compatiblecarriers, and optionally other therapeutic agents. In preferredembodiments of the present invention the Payload(s) is not covalentlyattached to the molecular network of the invention.

As used herein the terms “multi-photon responsive element” and“multi-photon responsive moiety” have equivalent meanings.

As used herein, the term “tier” refers to the a generation, or a level,in a dendritic structure.

As used herein, the term “amplifying sensitivity”, or “amplifying”,refers to where the number of Payload molecules released is greater thanthe number of covalent bonds cleaved during the release step. The term“amplifying” may also refer to the where the absorption of multi-photonirradiation that results in the cleavage of a multi-photon responsiveelement, causes in turn the cleavage of self-immolative oligomerscovalently attached to the multi-photon responsive element. The term“amplifying sensitivity” also refers to, in the area of detection, wherethe signal resulting from two-photon irradiation is substantiallystronger than can be accounted for by the irradiation itself.

As used herein, the term “particle” refers to small particles assembledaccording to embodiments of the present invention. The term “particle”may refer to nanoparticles or microparticles or both.

The term “microparticle”, as used herein, generally refers to a particlebetween 0.1 and 200 micrometers in size.

The term “nanoparticle”, as used herein, generally refers to discretestructures that are at least under 200 nm in diameter, in other words,significantly less than the diameter of a capillary. (See, e.g., Singh,R., J. W. Lillard Jr., Exper Mol Path 86 (2009) 215-223.) The term“nanoparticle” may also refer to particles that are between 1 nm and 100nm in diameter. Some of the novel properties associated withnanoparticles, which differentiate them from bulk materials, isgenerally associated with their size being less than 100 nm.

Microparticles and/or nanoparticles can be formed by a wide variety ofmeans and with widely varying compositions. Examples include hydrogelssuch as acrylamide micelle polymerization. The may also be created fromsuch diverse substances as poly(D,L) lactides; poly (lactic acid)(“PLA”); poly (D,L glycolide) (“PLG”); poly(lactide-co-glycolide)(“PLGA”); and poly-cyanoacrylate (“PCA”) (Singh, R., J. W. Lillard Jr.,Exper Mol Path 86 (2009) 215-223). Microparticles and/or nanoparticlescan also be created from various forms of micelles/liposomes; suchmicelles/liposomes can be assembled via emulsions or through a processof depositions. Acrylamide hydrogels, such as ones made fromN-isopropylacrylamide (NIPAAm) and acrylamide (AAm) have been madeincorporating gold-gold sulfide nanoshells designed to strongly absorbnear-infrared light, e.g., wavelengths between 800 and 1200 nm. Whenthese nanoparticles are irradiated, the temperature is increased,causing in turn the release of associated molecular cargo (Sershen S R,Westcott S L, Halas N J, West J L; Temperature-sensitivepolymer-nanoshell composites for photothermally modulated drug delivery;J Biomed Mater Res. (2000) 51(3):293-98. Examples of microparticlesand/or nanoparticles also include dendrimers (see, e.g., Cheng, Y., J.Wang, T. Rao, X. He, T. Xu, T.; Pharmaceutical applications ofdendrimers: promising nanocarriers for drug delivery; Front. Biosci. 13(2008) 1447-1471). Types of microparticles and/or nanoparticles havealso been described in PCT US2007/006844.

Microparticles and/or nanoparticles can also be formed according to theteachings of the present invention.

The term “molecular network”, or the term “molecular cage”, as usedherein refers to a covalently bound network comprising polymers thatcomprise polyacrylamide, or its equivalent, and further may comprisePEG, or its equivalent, and where these polymer(s), together with PEG orits equivalent, are covalently attached to self-immolative oligomer(s)which are covalently attached to multi-photon responsive element(s).

The terms “administration” or “administering” refer to a method ofincorporating a compound into the cells or tissues of an animal,preferably a mammal, in order to treat or prevent an abnormal condition.When the composition of the invention is provided in combination withone or active agents, the terms “administration” or “administering”include sequential or concurrent introduction of the composition withthe other agent(s). For cells harbored within the organism, manytechniques exist in the art to administer compounds, including (but notlimited to) oral, injection, parenteral, dermal, and aerosolapplications.

Example 1

Microparticles have previously been developed where one triggering eventleads to the cleavage of one bond (signal-to-output ratio equals 1).(See, e.g., Cohen, Almutairi et al., supra] In some embodiments of theinstant invention, however, a single triggering event leads to thenumber of cleaved bonds equal to the number of branches (in the case ofdendritic crosslinker), or to the number of monomer units (in the caseof linear polymeric crosslinkers), thus providing a greatly amplifiedresponse. To demonstrate the feasibility of this approach a firstgeneration dendritic crosslinker is compared with a second generationdendritic crosslinker in Example 1.

The synthesis of crosslinker 1 is outlined in FIG. 7. Compound 4 can besynthesized from commercially available 2,6-bishydroxymethyl-p-cresol,2, by first protecting the benzylic alcohol groups of 2 withtert-butylsilylchloride (TBSCl) to give compound 3, and then reacting itwith p-nitrophenyl-chloroformate in the presence of catalytic amount of4-dimethylaminopyridine (DMAP). Compound 5 can be obtained fromcommercially available N,N′-dimethyl-ethylenediamine and di-t-butyldicarbonate (Boc anhydride).

Reacting compound 4 with compound 5 produces compound 6. Removal of theprotecting groups with Amberlyst™ followed by reaction withp-nitrophenyl-chlorophormate yields compound 8. Compound 9 can beobtained by reaction of 4 with 5 followed by removal of the Bocprotecting group. Reacting compounds 8 and 9 yields compound 10.Deprotection of the hydroxyl groups of compound 10 followed byconversion into p-nitrophenylcarbonates and reaction with compound 11yields compound 12. A triggering group can be attached by reactingcompound 12 with the corresponding p-nitrophenylcarbonate to furnish thedesired self-immolative dendritic cross-linker 1.

Bromo-coumarin 13, a well established class of two-photon protectinggroups, was used as a two-photon photoremovable protecting group.

Example 2

Monomer 1 was synthesized largely according to a previously publishedprocedure (Amir, R. J.; Pessah, N.; Shamis, M.; Shabat, D. Angew. Chem.,Int. Ed. 2003, 42, 4494-4499) with slight modifications.4,5-Dimethoxy-2-nitrobenzyl alcohol (Patchornik, A; Amit, B.; Woodward,R. B. J. Am. Chem. Soc. 1970, 92, 6333-6335) was chosen despite its lowtwo-photon uncaging cross-section (0.01 GM) (Aujard, I, Benbrahim, C,Gouget, M, Ruel, 0, Baudin, J. B., Neveu, P, Chem. Eur. J. 2006, 12,6865-6879) compared to 4-bromo-coumarins (1 GM) (Furuta, T, Wang, S. S.H., Dantzker, J. L., Dore, T. M., Bybee, W. J., Callaway, E. M., Denk,W. Tsien, R. Y., Proc. Natl. Acad. Sci. U.S.A., 1999, 96, 1193-1200) orfluorene-based systems (5 GM) (Gug, S, Bolze, F. Specht, A. Bourgogne,C. Goeldner, M. Nicour, J.-F., Angew. Chem. Int. Ed., 2008, 47,9525-9529), because it is well-studied and readily available, making ita good proof-of-concept photolabile group.

Monomer 1 was copolymerized with adipoyl chloride to yield a regularcopolymer. The low molecular weight oligomers were removed by repeatedprecipitation of the crude polymer with cold ethanol, yielding the finalproduct with a molecular weight of 65,000 Da and PDI of 1.54(characterized by GPC relative to polystyrene standards) with 44% yield.

Cleavage of the multi-photon responsive element triggering groups byirradiation at 350 nm and 750 nm, via one and two-photon processesrespectively, was monitored by observing the changes in the absorbancespectrum of polymer 2 in acetonitrile/H₂O (9/1). Upon light exposure,the peak at 346 nm, corresponding to 4,5-dimethoxy-2-nitrobenzylcarbamate decreased, while a new peak at 400 nm appeared, correspondingto the cleaved 4,5-dimethoxy-2-nitrosobenzaldehyde. The absorptionspectrum remained unchanged after 15 minutes of irradiation with 350 nmlight, indicating complete deprotection, while it was necessary toirradiate the system for 5 hours at 750 nm to observe changes in theabsorption spectrum, consistent with the low two-photon uncagingcross-section of 4,5-dimethoxy-2-nitrobenzyl group.

The degradation of polymer 2 was studied by GPC and proton NMR inacetonitrile/water solutions. The polymer solutions were exposed to UVlight (350 nm) for various periods of time and incubated at 37° C.Samples were removed and analyzed. The degree of polymer degradationshowed strong dependence on the irradiation time (FIG. 13). The initialdrop in molecular weight in the first few minutes after UV irradiationis likely to be mostly due to the loss of the triggering groups, whilefurther reduction in molecular weight is due to the cleavage of thepolymer backbone as a result of cyclization and elimination reactionswithin the self-immolative monomer unit. The difference in thedegradation degree is especially evident in the samples irradiated for 5and 15 minutes: more multi-photon responsive element triggering groupsare cleaved. Consequently, the polymer chains degrade into smallerfragments. Although the estimated molecular weights of the fragmentslevel off at 20,000 Da the molecular weight of monomer 1 (m/z=544.19)was estimated by GPC to be 3,500 Da, therefore these fragments may beoligomers. Notably, only a small portion of all the multi-photonresponsive element triggering groups need to be cleaved to induce areduction in the molecular weight of the polymer.

The proton NMR of polymer 2 before irradiation in CDCl₃ showed all thecharacteristic peaks and splittings. As expected, in CD₃CN/D₂O the NMRpeaks broadened. Upon irradiation and incubation at 37° C. for 18 hoursthe NMR peaks corresponding to the benzylic protons of4,5-dimethoxy-2-nitrobenzyl group disappeared and the remaining peaksshifted accordingly. Sharper monomer peaks, indicating the presence ofurea and cresol emerged, overlapping with broader polymer and oligomerpeaks.

The cyclization of the diamine linker has been shown to be therate-determining step of the self-immolation within the quinine-methideunit, and it has been shown to accelerate in the presence oftriethylamine (Amir, R. J.; Pessah, N.; Shamis, M.; Shabat, D.; Angew.Chem., Int. Ed. (2003) 42, 4494-99). Thus, we measured polymerdegradation in the presence of triethylamine (FIG. 15) and observed anincrease in the rate of the polymer degradation (FIG. 14).

Two-photon irradiation of polymer 2 for 5 hours showed a similar degreeof degradation as 5 minute one-photon irradiation (FIG. 16).

Example 3

To evaluate the properties of the new polymer for controlledlight-triggered release, nanoparticles were formulated by the singleemulsion method (FIG. 17), encapsulating the small hydrophobic moleculedye Nile Red. This small molecule was chosen because of its excellentphotostability. The Z-average diameter of the nanoparticles was 170 nmand PDI=0.191, as determined by dynamic light scattering (DLS).

The release of the Nile Red Payload upon irradiation was observed byfluorescence spectroscopy. Nanoparticles were redispersed in PBS pH 7.4and the fluorescence intensity of the suspension was recorded. Afterirradiation with 350 nm light for 1 min, the fluorescence intensitydropped by 67%, indicating burst release of the dye from thenanoparticles into a more polar medium (FIGS. 17 and 18). On the otherhand, a suspension of nanoparticles that was not irradiated exhibitedunchanged fluorescence intensity over several days. Interestingly,prolonged irradiation of nanoparticles did not result in a further dropof fluorescence signal.

Further degradation of nanoparticles of 2 after UV irradiation wasobserved by DLS at 37° C. in PBS buffer at pH 7.4 and pH 10. Noparticles were detected after 4 days of incubation at pH 10 while in pH7.4 the particles degraded within 10 days.

We also explored the possibility of triggering the release of Nile Redby NIR light through two-photon absorption. The suspension ofnanoparticles in PBS pH 7.4 was irradiated at 750 nm for 20 minintervals followed by 10 min of incubation at 37° C. A gradual decreasein the fluorescence intensity of Nile Red was observed during the 4hours of irradiation (FIG. 19).

The observation of burst release of Nile Red upon UV irradiation whilethe polymer degradation is slower suggests the possibility of asecondary mechanism of release. A change in hydrophobicity of theparticles upon cleavage of the multi-photon responsive elementtriggering group may be involved. The rapid and efficient unmasking of alarge number of the secondary amino groups may make the particlesrapidly more permeable to water. This may explain the rapid release ofNile Red upon UV irradiation. However, the two-photon unmasking processis much less efficient which could explain the slower Nile Red releasein the NIR two-photon regime. Notably, the final degradation of thenanoparticles is an important property for in vivo biologicalapplications that require materials to degrade into easily excretablefragments.

To rule out the possibility of spontaneous release caused by simplecavitation, poly(lactic-co-glycolic acid) (PLGA) nanoparticlesencapsulating Nile Red were formulated by the same method and exposed toUV and NIR light in the same fashion. As expected, no release of NileRed was observed in this case.

Example 4

More efficient two-photon uncaging coumarin-based groups are used inplace of the nitrobenzyl of Example 2, supra, group to make the systemmore sensitive to NIR light increase their utility. When irradiated withnear infrared radiation, the nanoparticles release their payload.

Example 5

The versatile design of the present invention allows the multi-photonresponsive element triggering group to be sensitive to internal orremote stimuli. A polymer was synthesized with a multi-photon responsiveelement having increased efficiency, specifically 4-bromo-coumarin (1GM) shown in FIG. 20. Polymers made with this multi-photon responsiveelement degraded rapidly upon exposure to 800 nm light. Nanoparticlesmade from this polymer are being formulated and characterized.

Example 6

The self-immolative polymer shown in FIG. 21 was synthesized as shown inFIG. 22 and formulated into nanoparticles.

Its degradation properties are being characterized; the rate-determiningstep in the mechanism of this type of polymer degradation is thecyclization to form the cyclic urea.

Example 7

Additional two-photon-labile crosslinkers such as the one in shown inFIG. 23 were developed. This crosslinker was used to form nanoparticlesand gels. The method used to synthesize this crosslinker is shown inFIG. 24. Degradation of this crosslinker was demonstrated with near-UVlight as well as 800 nm light. Its cleavage was demonstrated by theresults depicted in FIG. 25.

These new crosslinkers were used to formulate two-photon-labilecrosslinked hydrogels using PEG-Acrylate initiated with TMEDA, which areshown in FIG. 26. These hydrogels incorporating multi-photon responsiveelements were formulated by using 480 mg of PEG-Acrylate plus 1 mol % of(PEG-Diacrylate/multi-photon) crosslinker plus 200 μl of buffer(±Rhodamine dextran) plus 20 μl of 50% APS. Formation of the gel wasinitiated using 20 μl of TMEDA and continued at 37° C. for 24 hrs.

These hydrogels were demonstrated to have a low toxicity using twodifferent assays. The results of these assays are shown in FIG. 27.Growth of cells was tested in the presence of ˜30 mg of the hydrogelsformulated with multi-photon-responsive elements in 2 ml of media(6-well dishes). The pieces of gel were treated or untreated withexposure to 20 minutes of UV. The multi-photon-labile gel material wasalso tested in the MTT-toxicity assay.

Example 8

Materials with polymeric backbones are synthesized wherein the backboneshave been molecularly engineered to incorporate units capable ofabsorbing multi-photon radiation simultaneously and initiating a cascadeof changes within the higher order assembly. FIG. 28 depicts one suchpolymer based on a polyester backbone that will disassemble upondeprotection of the hydroxyl protecting group. This polymer will undergobackbiting upon deprotection of the pendant alcohol. The alcohols areprotected with a variety of protecting groups. In the initialexperiments, model protecting groups such as those used elsewhere areused. Other protecting groups may be used.

The proposed synthesis of the proposed polyester backbone is shown inFIG. 29. These polyesters will be synthesized from the commerciallyavailable butyrolactone 4-(hydroxymethyl)-1,3-dioxolan-2-one via aring-opening polymerization using distannoxane catalysts.

This backbone may be used with the methods and compounds of the presentinvention.

Example 9

Materials with polymeric backbones are synthesized wherein the backboneshave been molecularly engineered to incorporate units capable ofabsorbing multi-photon radiation simultaneously and initiating a cascadeof changes within the higher order assembly. FIG. 30 depicts three suchpolymers based on a polyester and/or polycarbonate backbones that willdisassemble upon deprotection of the hydroxyl protecting group and thusmay be used with the methods and compounds of the present invention.

Example 10

Nanoparticles of the present invention are synthesized in the presenceof stem cell differentiation agents. They are administered to a patientin need of such treatment and disrupted in the immediate vicinity of anorgan that has failed or is performing sub-optimally and is in need ofstem cell therapy.

Example 11

Nanoparticles of the present invention are loaded with non-infectiousviral particles, DNA associated with a viral infection or associatedwith a disease, proteins associated with a viral infection or associatedwith a disease, and/or adjuvants. They are used to optimize cytotoxic Tlymphocyte responses and antibody responses by sequestering vaccineand/or immunogen components until they reach a location within apatient's body where the components will produce their maximum effect.They are released at that location according to the methods of thepresent invention.

Example 12

A combination of nanoparticles are synthesized according to the presentinvention the Payload(s) in one set comprises a luminescent enzyme andthe Payload(s) in the other set comprises the enzyme's substrate. Asmall number of multi-photon absorption events results in a significantburst of luminescence as the enzyme and substrate are united.

Example 13

Nanoparticles are synthesized according to the present invention wherePayload(s) comprises antibodies. Multi-photon absorption events canreleases multiple antibodies capable of binding multiple antigens at thepoint of release.

Example 14

Encapsulation of viral particles, DNA, proteins, and adjuvants, in thesame nanocarrier may help to realize optimal cytotoxic T lymphocytes(CTLs) responses and antibody responses by sequestering the vaccinecomponents until they reach the target cell, delivering them to aparticular class of cells, and/or aiding in their presentation via thedesired pathway. One disease where the treatment will benefit from suchtechnology is Herpes Simplex Virus-2.

HSV-2 causes a lifelong, persistent infection resulting in recurrentgenital lesions. Transmission of the virus at birth also can causedevastating disease in the newborn. Of greatest importance worldwide,particularly in developing countries, is that genital HSV-2 infectionsignificantly increases the risk of acquisition and transmission ofHIV-1. Although antivirals are available, the lifelong persistence ofthis virus provides a strong impetus for the development of an effectivevaccine.

Viral components included in a nanoparticle synthesized according to thepresent invention will include whole-inactivated virus as well as DNAplasmids encoding three specific HSV-2 genes. The first benefit of thisapproach is that whole-inactivated virus generates broader neutralizingantibody responses than single HSV-2 glycoprotein D subunit currently invaccine trials. The DNA plasmids will comprise the HSV-2 gene encodingglycoprotein D as well as two genes that are highly conserved essentialviral nonstructural proteins, viral DNA polymerase and helicase/primase.These genes will be excellent targets in infected cells for “primed” CD8or CD4 T cells since immunization with the DNA will also generate CD4and CD8 T-cell responses. Delivery of these antigens by DNA in thenanoparticle of the present invention, should bypass viral immunoevasivemechanisms, and the high T cell responses elicited may provide improvedimmunity over the natural infection.

A nanocarrier may be developed from acetalated-dextran containing DNAplasmids encoding HSV-2 glycoprotein D, DNA polymerase, andhelicase/primase genes; formalin-inactivated HSV-2 virions; and anadjuvant such as MPL. Acetalated-dextran may be used in the DoubleEmulsion Condensation method to entrap different combinations of DNAplasmids and formalin-inactivated HSV-2 together with the adjuvant. Theprotective and immunological responses for each nanocarrier vaccinepayload will be assessed.

Methods

BALB/c female mice will be immunized by subcutaneous injection twice, onweeks 0 and 4, with nanoparticle vaccines prepared according to thepresent invention or glycoprotein D subunit/MPL/alum vaccine, and theninjected subcutaneously with 3 mg of medroxyprogesterone acetate(Depo-Provera) on days 21 and 27 after the last immunization tosynchronize the estrus cycle and to increase susceptibility to vaginalHSV infection; on day 28, mice will be intravaginally challenged with10×LD50 of HSV-2 strain G. Mice will be scored daily for 21 days for theseverity of vaginal inflammation from 0 (no vaginitis), 1 (mild swellingor redness only), 2 (moderate swelling or erosions), 3 (severe genitalmaceration), or 4 (central nervous system involvement or death). Vaginalswab samples will be collected on days 1 to 7 post challenge to confirmviral replication and measure shedding. To evaluate immune responses, 3weeks after the first and second immunization, several mice in eachgroup will be bled and then sacrificed for isolation of splenocytes todetermine levels of antibodies and primary T cell subsets, respectively,elicited. At 5 days after challenge, several mice in each group will besacrificed to measure the recall responses.

Positive results from the BALB/c mouse tests will be more rigorouslyevaluated and compared with the HSV-2 glycoprotein D subunit/MPL/alumvaccine in the guinea pig model of genital HSV-2 infection. Guinea pigsdevelop external genital lesions that can be graded according toseverity and are analogous to humans, in that the virus reactivatescausing recurrent genital lesions.

Female Hartley guinea pigs will be immunized by bilateral injection inthe quadriceps (4 weeks apart) with the optimal nanoparticlecombinations or with HSV-2 glycoprotein D in MPL/alum. Four weeks afterthe last boost, anesthetized guinea pigs will be intravaginallychallenged with 5.7 log 10 PFU of HSV-2 strain G. Animals will be scoreddaily for lesion development based on a severity scale of 0) no disease,0.5) slight erythema/swelling only, 1) erythema/swelling only, 2) singleor a few small lesions, 3) large or fused vesicles, and 4) ulceratedlesions. Viral shedding in the vagina will be measured on days 2, 5, 7,and 10 postchallenge and recurrence of vaginal disease will be assessedthrough day 90 postchallenge. At the conclusion of the experiment, DNAwill be purified from the lumbrosacral ganglia from each guinea pig andused for real-time PCR quantification of latent HSV-2 DNA.

Example 15

A polymer bearing boronic esters, shown in FIG. 21, was synthesized.Such a compound is sensitive to hydrogen peroxide. Cleavage of thispolymer by hydrogen peroxide was demonstrated. Higher hydrogen peroxideconcentrations in vivo are known to be associated with inflammatory andcardiovascular diseases.

Nanoparticles and microparticles of these polymers will be formulatedand their efficacy in a variety of biomedical applications will bedemonstrated.

Thus, embodiments of the present invention exist wherein themulti-photon responsive element may be replaced by a hydrogen peroxideresponsive element.

1. A composition comprising a multi-photon responsive element covalentlylinked to a self-immolative backbone subunit.
 2. The composition ofclaim 1 wherein said multi-photon responsive element is a two-photonresponsive element.
 3. The composition of claim 2 wherein saidmulti-photon responsive unit is a bromo-coumarin group.
 4. Thecomposition of claim 1 wherein said composition further comprises amolecular network comprising polyacrylamide polymers or PEG polymers. 5.(canceled)
 6. (canceled)
 7. The composition of claim 4 wherein saidcomposition further comprises a Payload, comprising a pharmaceuticalagent, stem cell differentiation agents, immunogen or antibodies. 8.(canceled)
 9. The composition of claim 1 wherein said self-immolativebackbone subunit is an oligomer comprising comprises the structure

or derivatives thereof. 10.-17. (canceled)
 18. A method delivering aPayload to a tissue comprising the steps of: synthesizing a particlewherein said particle further comprises a multi-photon responsiveelement, a self-immolative backbone subunit, a molecular network, and aPayload; administering said particle to said tissue; and irradiatingsaid tissue comprising said particle with electromagnetic radiation;wherein said particle is disrupted in situ following absorption of saidelectromagnetic radiation.
 19. The method of claim 18, wherein saidelectromagnetic radiation is near infrared red light or ultravioletlight.
 20. (canceled)
 21. The method of claim 18, wherein said methodprovides an amplified response to said electromagnetic radiation. 22.The method of claim 18, wherein said Payload comprises a pharmaceuticalagent, stem cell differentiation agents, immunogen or antibodies.23.-25. (canceled)
 26. A method for amplifying sensitivity toelectromagnetic radiation by irradiating the composition of claim 1.27.-43. (canceled)
 44. A method for amplifying sensitivity toelectromagnetic radiation by irradiating the composition of claim
 2. 45.A method for amplifying sensitivity to electromagnetic radiation byirradiating the composition of claim
 3. 46. A method for amplifyingsensitivity to electromagnetic radiation by irradiating the compositionof claim
 4. 47. A method for amplifying sensitivity to electromagneticradiation by irradiating the composition of claim
 7. 48. A method foramplifying sensitivity to electromagnetic radiation by irradiating thecomposition of claim 9.